Nucleic acid molecules encoding glutx and uses thereof

ABSTRACT

The invention concerns the human gene encoding GLUTX, a glucose transporter. GLUTX nucleic acid and polypeptides, as well as molecules which increase or decrease expression or activity of GLUTX, are useful in the diagnosis and treatment of disorders associated with aberrant hexose transport.

BACKGROUND OF THE INVENTION

[0001] A number of mammalian glucose (hexose) transporters (GLUTs) havebeen identified. High affinity GLUTs are found in nearly every tissue. Alow affinity GLUT (GLUT-2) is expressed in tissues which are associatedwith high glucose flux (e.g., intestine, kidney, and liver). It isthought that the level of expression of high affinity GLUTs influencesthe rate of glucose uptake. It is also thought that the expression ofvarious GLUTs is regulated by glucose and various hormones (Thorens, Am.J. Physiol. 270 (Gastrointest. Liver Physiol. 33:G541-G553, 1996). HumanGLUT-1 is described by Mucckler et al. (Science 229:941, 1985). HumanGLUT-2 is described by Fukumoto et al. (Proc. Nat'l Acad. Sci. USA264:776, 1989). Human GLUT-3 is described by Keller et al. (J. Biol.Chem. 264:18884, 1989). Human GLUT-4 is described by Fukumoto et al. (J.Biol. Chem. 264:7776, 1989). Human GLUT-5 is described by Kayano et al.(Nature 377:151, 1995).

SUMMARY OF THE INVENTION

[0002] The invention described herein relates to the discovery andcharacterization of a cDNA encoding GLUTX, a human glucose transporterprotein. The nucleotide sequence of a cDNA encoding GLUTX is shown inFIG. 1. The deduced amino acid sequence of GLUTX is shown in FIG. 2.GLUTX is predicted to include 12 transmembrane domains. The firsttransmembrane domain extends from about amino acid 52 (intracellularend) to about amino acid 71 (extracellular end). The secondtransmembrane domain extends from about amino acid 108 (extracellularend) to about amino acid 128 (intracellular end). The thirdtransmembrane domain extends from about amino acid 141 (intracellularend) to about amino acid 159 (extracellular end). The fourthtransmembrane domain extends from about amino acid 166 (extracellularend) to about amino acid 189 (intracellular end). The fifthtransmembrane domain extends from about amino acid 204 (intracellularend) to about amino acid 221 (extracellular end). The sixthtransmembrane domain extends from about amino acid 233 (extracellularend) to about amino acid 252 (intracellular end). The seventhtransmembrane domain extends from about amino acid 317 (intracellularend) to about amino acid 338 (extracellular end). The eighthtransmembrane domain extends from about amino acid 355 (extracellularend) to about amino acid 375 (intracellular end). The ninthtransmembrane domain extends from about amino acid 383 (intracellularend) to about amino acid 404 (extracellular end). The tenthtransmembrane domain extends from about amino acid 413 (extracellularend) to about amino acid 437 (intracellular end). The eleventhtransmembrane domain extends from about amino acid 449 (intracellularend) to about amino acid 472 (extracellular end). The twelfthtransmembrane domain extends from about amino acid 481 (extracellularend) to about amino acid 499 (intracellular end). GLUTX nucleic acidsand polypeptides, as well as molecules which increase or decreaseexpression or activity of GLUTX, are useful in the diagnosis andtreatment of disorders associated with aberrant hexose transport.

[0003] GLUTX protein has some sequence similarity to a number of knownglucose transporters (FIG. 3).

[0004] The invention features isolated nucleic acid molecules (i.e., anucleic acid molecule that is separated from the 5′ and 3′ codingsequences with which it is immediately contiguous in the naturallyoccurring genome of an organism, also referred to as a recombinantnucleic acid molecule) that encodes a GLUTX polypeptide. Within theinvention are polypeptides having the sequence of SEQ ID NO:2 or encodedby nucleic acid molecules having the sequence shown in SEQ ID NO:1.However, the invention is not limited to nucleic acid molecules andpolypeptides that are identical to those SEQ ID Nos. For example, theinvention includes nucleic acid molecules which encode splice variants,allelic variants or mutant forms of GLUTX as well as the proteinsencoded by such nucleic acid molecules.

[0005] Also within the invention are nucleic acid molecules thathybridize under stringent conditions to a nucleic acid molecule havingthe sequence of SEQ ID NO:1. Such molecules include, for example,nucleic acid molecules encoding allelic variants of GLUTX or mutantforms of GLUTX. As described further below, molecules that aresubstantially identical to those of SEQ ID Nos. 1 and 2 are alsoencompassed by the invention.

[0006] The term “substantially pure” as used herein in reference to agiven compound (e.g., a GLUTX polypeptide) means that the compound issubstantially free from other compounds, such as those in cellularmaterial, viral material, or culture medium, with which the compound mayhave been associated (e.g., in the course of production by recombinantDNA techniques or before purification from a natural biological source).When chemically synthesized, a compound of the invention issubstantially pure when it is substantially free from the chemicalcompounds used in the process of its synthesis. Polypeptides or othercompounds of interest are substantially free from other compounds whenthey are within preparations that are at least 60% by weight (dryweight) the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. Purity can be measured by anyappropriate standard method, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

[0007] Where a particular polypeptide or nucleic acid molecule is saidto have a specific percent identity to a reference polypeptide ornucleic acid molecule of a defined length, the percent identity isrelative to the reference polypeptide or nucleic acid molecule. Thus, apeptide that is 50% identical to a reference polypeptide that is 100amino acids long can be a 50 amino acid polypeptide that is completelyidentical to a 50 amino acid long portion of the reference polypeptide.It might also be a 100 amino acid long polypeptide which is 50%identical to the reference polypeptide over its entire length. ofcourse, many other polypeptides will meet the same criteria. The samerule applies for nucleic acid molecules.

[0008] For polypeptides, the length of the reference polypeptidesequence will generally be at least 16 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids, 50 amino acids, or 100 amino acids. Fornucleic acids, the length of the reference nucleic acid sequence willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably at least 100 nucleotides (e.g., 150, 200, 250, or 300nucleotides).

[0009] In the case of polypeptide sequences that are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, onservative substitutions for thereference sequence. onservative substitutions typically includesubstitutions ithin the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0010] Sequence identity can be measured using sequence analysissoftware (e.g., the Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705 with the default parameters asspecified therein.

[0011] The BLAST programs, provided as a service by the National Centerfor Biotechnology Information (http://www.ncbi.nlm.nih.gov), are veryuseful for making sequence comparisons. The programs are described indetail by Karlin et al., (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990and 90:5873-7, 1993) and Altschul et al., (Nucl. Acids Res.25:3389-3402, 1997) and are available on the internet at:http://www.ncbi.nlm.nih.gov.

[0012] The invention also features a host cell that harbors an isolatednucleic acid molecule encoding GLUTX (either alone or in conjunctionwith a heterologous polypeptide, such as a detectable marker) or anucleic acid vector that contains a sequence encoding GLUTX (again, withor without a heterologous polypeptide). The vector can be an expressionvector, and the expression vector can include a regulatory element. Anantibody that specifically binds a GLUTX polypeptide is also within thescope of the present invention and is useful, for example, to detectGLUTX in a biological sample or to alter the activity of GLUTX. Forexample, GLUTX can be detected in a biological sample by contacting thesample with an antibody that specifically binds GLUTX under conditionsthat allow the formation of a GLUTX-antibody complex and detecting thecomplex, if present, as an indication of the presence of GLUTX in thesample. The use of an antibody in a treatment regime, where it can alterthe activity of GLUTX, is discussed further below.

[0013] An antibody of the invention can be a monoclonal, polyclonal, orengineered antibody that specifically binds GLUTX (as described morefully below). An antibody that “specifically binds” to a particularantigen, for example, a GLUTX polypeptide of the invention, will notsubstantially recognize or bind to other molecules in a sample, e.g., abiological sample, that includes GLUTX.

[0014] Given that an object of the present invention is to alter theexpression or activity of GLUTX in vivo, a pharmaceutical compositioncontaining, for example, an isolated nucleic acid molecule encodingGLUTX (or a fragment thereof), a nucleic acid molecule that is antisenseto GLUTX (i.e., that has a sequence that is the reverse and complementof a portion of the coding strand of a GLUTX gene), a GLUTX polypeptide,or an antibody, small molecule, or other compound that specificallybinds a GLUTX polypeptide is also a feature of the invention.

[0015] The discovery and characterization of GLUTX and the polypeptideit encodes makes it possible to determine whether a given disorder isassociated with aberrant expression of GLUTX (either at thetranscriptional or translational level) or activity of GLUTX. Forexample, one can diagnose a patient as having a disorder associated withaberrant expression of GLUTX by measuring GLUTX expression in abiological sample obtained from the patient. An increase or decrease inGLUTX expression in the biological sample, compared with GLUTXexpression in a control sample (e.g., a sample of the same tissuecollected from one or more healthy individuals) indicates that thepatient has a disorder associated with aberrant expression of GLUTX.Similarly, one can diagnose a patient as having a disorder associatedwith aberrant activity of GLUTX by measuring GLUTX activity in abiological sample obtained from the patient. An increase or decrease inGLUTX activity in the biological sample, compared with GLUTX activity ina control sample, indicates that the patient has a disorder associatedwith aberrant activity of GLUTX. The techniques required to measure geneexpression or polypeptide activity are well known to those of ordinaryskill in the art.

[0016] In addition to diagnostic methods, such as those described above,the present invention encompasses methods and compositions for typingand evaluating the prognosis of patients suffering from a disorderassociated with aberrant activity or expression of GLUTX. The inventionalso encompasses methods and compositions for selecting an appropriatean treatment for disorders associated with inappropriate expression ofGLUTX or inappropriate activity of GLUTX. The invention also includescompositions and methods for assessing the effectiveness of suchtreatments. For example, the nucleic acid molecules of the invention canbe used as probes to classify cells in terms of their level of GLUTXexpression and as primers for diagnostic PCR analysis which can be usedto detect mutations, allelic variations, and regulatory defects in theGLUTX gene. Similarly, those of ordinary skill in the art can useroutine techniques to identify inappropriate activity of GLUTX, whichcan be observed in a variety of forms. Diagnostic kits for the practiceof such methods are also provided.

[0017] The invention further encompasses transgenic animals that expressGLUTX and recombinant “knock-out” animals that fail to express GLUTX.These animals can serve as new and useful models of disorders in whichGLUTX is misexpressed.

[0018] The invention also features antagonists and agonists of GLUTXthat can inhibit or enhance, respectively, one or more of the biologicalactivities of GLUTX, e.g., the ability to act as a transporter forcertain sugars. Suitable antagonists can include small molecules (i.e.,molecules with a molecular weight below about 500), large molecules(i.e., molecules with a molecular weight above about 500), antibodiesthat specifically bind and “neutralize” GLUTX (as described below), andnucleic acid molecules that interfere with transcription or translationof GLUTX (e.g., antisense nucleic acid molecules and ribozymes).Agonists of GLUTX also include small and large molecules, and antibodiesother than neutralizing antibodies.

[0019] The invention features methods and compositions useful foridentifying antagonists and agonists of a GLUTX biological activity.These methods entail measuring the activity of GLUTX in the presence andabsence of a test compound.

[0020] The invention also features molecules that can increase ordecrease the expression of GLUTX (e.g., by altering transcription ortranslation). Small molecules (as defined above), large molecules (asdefined above), and nucleic acid molecules (e.g., antisense and ribozymemolecules) can be used to inhibit the expression of GLUTX. Other typesof nucleic acid molecules (e.g., molecules that bind to GLUTX negativetranscriptional regulatory sequences) can be used to increase theexpression of GLUTX.

[0021] Compounds that modulate the expression of GLUTX in a cell can beidentified by comparing the level of expression of GLUTX in the presenceof a selected compound with the level of expression of GLUTX in theabsence of that compound. A difference in the level of GLUTX expressionindicating that the selected compound modulates the expression of GLUTXin the cell. A comparable test for compounds that modulate the activityof GLUTX can be carried out by comparing the level of GLUTX activity inthe presence and absence of the compound. Thus, the in

[0022] The invention features methods and compositions useful foridentifying compounds which modulate GLUTX expression. These methodsentail measuring the expression of GLUTX (at the transcriptional ortranslational level) in the presence and absence of a test compound.

[0023] Patients who have a disorder mediated by abnormal GLUTX activitycan be treated by administration of a compound that alters theexpression of GLUTX or the activity of GLUTX. When the objective is todecrease expression or activity, the compound administered can be aGLUTX antisense oligonucleotide or an antibody, such as a neutralizingantibody, that specifically binds GLUTX, respectively.

[0024] The preferred methods and materials are described below inexamples which are meant to illustrate, not limit, the invention.Skilled artisans will recognize methods and materials that are similaror equivalent to those described herein, and that can be used in thepractice or testing of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In addition, the materials,methods, and examples are illustrative only and are not intended to belimiting.

[0026] Other features and advantages of the invention will be apparentfrom the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a depiction nucleotide sequence (SEQ ID NO:1) of humanGLUTX.

[0028]FIG. 2 is a depiction of the predicted amino acid sequence (SEQ IDNO:2) of human GLUTX.

[0029]FIG. 3 is comparison of the amino acid sequences of GLUTX (SEQ IDNO:2), GLUT1 (SEQ ID NO:3), GLUT2 (SEQ ID NO:4), GLUT3 (SEQ ID NO:5),GLUT4 (SEQ ID NO:6), and GLUT5 (SEQ ID NO:7).

[0030]FIG. 4 includes a series of plots predicting various structuralfeatures of GLUTX: alpha regions (Garnier-Robson), beta regions(Garnier-Robson), turn regions (Garnier-Robson), coil regions(Garnier-Robson), amphipathic alpha regions (Eisenberg), amphipathicbeta regions (Eisenberg), and flexible regions (Karplus-Schult). FIG. 4also includes plots of antigenicity index (Jameson-Wolf), surfaceprobability (Emini), and hydrophilicity (Kyte-Doolittle).

DETAILED DESCRIPTION

[0031] GLUTX is a glucose transporter which has some sequence similarityto members of the GLUT family. GLUTX is predicted to have 12transmembrane domains. The first transmembrane domain extends from aboutamino acid 52 (intracellular end) to about amino acid 71 (extracellularend). The second transmembrane domain extends from about amino acid 108(extracellular end) to about amino acid 128 (intracellular end). Thethird transmembrane domain extends from about amino acid 141(intracellular end) to about amino acid 159 (extracellular end). Thefourth transmembrane domain extends from about amino acid 166(extracellular end) to about amino acid 189 (intracellular end). Thefifth transmembrane domain extends from about amino acid 204(intracellular end) to about amino acid 221 (extracellular end). Thesixth transmembrane domain extends from about amino acid 233(extracellular end) to about amino acid 252 (intracellular end). Theseventh transmembrane domain extends from about amino acid 317(intracellular end) to about amino acid 333 (extracellular end). Theeighth transmembrane domain extends from about amino acid 355(extracellular end) to about amino acid 375 (intracellular end). Theninth transmembrane domain extends from about amino acid 383(intracellular end) to about amino acid 404 (extracellular end). Thetenth transmembrane domain extends from about amino acid 413(extracellular end) to about amino acid 437 (intracellular end). Theeleventh transmembrane domain extends from about amino acid 449(intracellular end) to about amino acid 472 (extracellular end). Thetwelfth transmembrane domain extends from about amino acid 481(extracellular end) to about amino acid 499 (intracellular end).

[0032] The GLUTX gene was identified as follows. A variety of public andproprietary sequence databases were searched using an approach designedto identify putative glucose transporters. This search led to theidentification of an EST which was thought likely to encode a portion ofa gene having some similarity to genes encoding previously identifiedglucose transporters. Two PCR primers (TGTTTCCTAGTCTTTGCTACA; SEQ IDNO:8 and TTGTTAAGGCCTTCCATT; SEQ ID NO:9) based on the sequence of theidentified EST were used to screen a human mixed tissue cDNA library.This screening resulted in the identification of a probe which was usedto screen the human mixed tissue cDNA library. This screening led to theidentification of a number of putative glucose transporter clones. Anumber of these clones were sequenced and ordered to arrive at acomplete sequence for GLUTX. The nucleotide sequence of GLUTX is shownin FIG. 1. The predicted amino acid sequence of GLUTX is shown in FIG.2.

[0033] The nucleic acid molecules of the invention and the polypeptidesthey encode (e.g., a GLUTX polypeptide or fragments thereof) can be useddirectly as diagnostic and therapeutic agents, or they can be used togenerate antibodies or identify small molecules that, in turn, areclinically useful. In addition, GLUTX nucleic acid molecules can be usedto identify the chromosomal location of GLUTX and as tissue-specificmarkers. Accordingly, expression vectors containing the nucleic acidmolecules of the invention, cells transfected with these vectors, thepolypeptides expressed by these cells, and antibodies generated, againsteither the entire polypeptide or an antigenic fragment thereof, areamong the preferred embodiments. These embodiments and some of theirclinical application are described further below.

[0034] I. Nucleic Acid Molecules Encoding GLUTX

[0035] The GLUTX nucleic acid molecules of the invention can be cDNA,genomic DNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded. In the event the nucleic acid molecule issingle-stranded, it can be either a sense or an antisense strand.Fragments of these molecules are also considered within the scope of theinvention, and can be produced, for example, by the polymerase chainreaction (PCR), or by treating a longer fragment (e.g., a full-lengthGLUTX gene sequence) with one or more restriction endonucleases.Similarly, a full-length GLUTX mRNA molecule, or a fragment thereof, canbe produced by in vitro transcription. The isolated nucleic acidmolecule of the invention can encode a fragment of GLUTX that is notfound as such in the natural state. Although nucleic acid moleculesencoding any given fragment of GLUTX are within the scope of theinvention, fragments that retain a biological activity of GLUTX arepreferred.

[0036] The nucleic acid molecules of the invention encompass recombinantmolecules, such as those in which a nucleic acid molecule (e.g., anisolated nucleic acid molecule encoding GLUTX, or a fragment thereof) isincorporated: (1) into a vector (e.g., a plasmid or viral vector), (2)into the genome of a heterologous cell, or (3) into the genome of ahomologous cell, at a position other than the natural chromosomallocation. Recombinant nucleic acid molecules, transgenic animals, anduses therefor are discussed further below.

[0037] The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. In addition, the nucleic acid molecules of theinvention are not limited to those that encode the amino acid residuesof the GLUTX polypeptide encoded by SEQ ID NO: 2; they can also includesome or all of the non-coding sequences that lie upstream or downstreamfrom a GLUTX coding sequence, a heterologous regulatory element, or asequence encoding a heterologous polypeptide (e.g., a reporter gene).Regulatory elements and reporter genes are discussed further below.

[0038] The nucleic acid molecules of the invention can be synthesized(for example, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, mouse, rat, guinea pig, cow, sheep, goat,horse, pig, rabbit, monkey, dog, or cat. Combinations or modificationsof the nucleotides within these types of nucleic acid molecules are alsoencompassed.

[0039] In the event the nucleic acid molecules of the invention encodeor act as antisense molecules, they can be used, for example, toregulate translation of GLUTX mRNA. Techniques associated with detectionof nucleic acid sequences or regulation of their expression are wellknown to persons of ordinary skill in the art, and can be used in thecontext of the present invention to diagnose or treat disordersassociated with aberrant GLUTX expression. However, aberrant expressionof GLUTX (or aberrant activity of GLUTX) is not a prerequisite fortreatment according to the methods of the invention; the molecules ofthe invention (including the nucleic acid molecules described here) areexpected to be useful in improving the symptoms associated with avariety of medical conditions regardless of whether or not theexpression of GLUTX (or the activity of GLUTX) is detectably aberrant.Nucleic acid molecules are discussed further below in the context oftheir clinical utility.

[0040] The invention also encompasses nucleic acid molecules that encodeother members of the GLUTX family (e.g., the murine homologue of GLUTX).Such nucleic acid molecules will be readily identified by the ability tohybridize under stringent conditions to a nucleic acid molecule encodinga GLUTX polypeptide (e.g., a nucleic acid molecule having the sequenceof SEQ ID NO:1). The cDNA sequence described herein (SEQ ID NO:1) can beused to identify these nucleic acids, which include, for example,nucleic acids that encode homologous polypeptides in other species,splice variants of the GLUTX gene in humans or other mammals, allelicvariants of the GLUTX gene in humans or other mammals, and mutant formsof the GLUTX gene in humans or other mammals.

[0041] The preferred class of nucleic acid molecules that hybridize toSEQ ID NO:l are nucleic acid molecules that encode human allelicvariants of GLUTX. There are two major classes of such variants: activeallelic variants, naturally occurring variants that have the biologicalactivity of GLUTX and non-active allelic variants, naturally occurringallelic variants that lack the biological function of GLUTX. Activeallelic variants can be used as an equivalent for a GLUTX protein havingthe amino acid sequence encoded by SEQ ID NO:1 as described hereinwhereas nonactive allelic variants can be used in methods of diseasediagnosis and as a therapeutic target.

[0042] The invention features methods of detecting and isolating suchnucleic acid molecules. Using these methods, a sample (e.g., a nucleicacid library, such as a cDNA or genomic library) is contacted (or“screened”) with a GLUTX-specific probe (e.g., a fragment of SEQ ID NO:1that is at least 17 nucleotides long). The probe will selectivelyhybridize to nucleic acids encoding related polypeptides (or tocomplementary sequences thereof). The term “selectively hybridize” isused to refer to an event in which a probe binds to nucleic acidmolecules encoding GLUTX (or to complementary sequences thereof) to adetectably greater extent than to nucleic acids encoding otherpolypeptides, particularly other types of transporter molecules (or tocomplementary sequences thereof). The probe, which can contain at least17 nucleotides (e.g., 18, 20, 25, 50, 100, 150, or 200 nucleotides) canbe produced using any of several standard methods (see, e.g., Ausubel etal., “Current Protocols in Molecular Biology, Vol. I,” Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., NY, 1989). For example,the probe can be generated using PCR amplification methods in whicholigonucleotide primers are used to amplify a GLUTX-specific nucleicacid sequence (for example, a nucleic acid encoding one of thetransmembrane domains) that can be used as a probe to screen a nucleicacid library and thereby detect nucleic acid molecules (within thelibrary) that hybridize to the probe.

[0043] One single-stranded nucleic acid is said to hybridize to anotherif a duplex forms between them. This occurs when one nucleic acidcontains a sequence that is the reverse and complement of the other(this same arrangement gives rise to the natural interaction between thesense and antisense strands of DNA in the genome and underlies theconfiguration of the double helix). Complete complementarity between thehybridizing regions is not required in order for a duplex to form; it isonly necessary that the number of paired bases is sufficient to maintainthe duplex under the hybridization conditions used.

[0044] Typically, hybridization conditions initially used to identifyrelated genes are of low to moderate stringency. These conditions favorspecific interactions between completely complementary sequences, butallow some non-specific interaction between less than perfectly matchedsequences to occur as well. After hybridization, the nucleic acids canbe “washed” under moderate or high conditions of stringency todissociate duplexes that are bound together by some non-specificinteraction (the nucleic acids that form these duplexes are thus notcompletely complementary).

[0045] As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (e.g., roomtemperature) using a solution containing a salt concentration that isequivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

[0046] In reactions where nucleic acids are hybridized, the conditionsused to achieve a given level of stringency will vary. There is not oneset of conditions, for example, that will allow duplexes to form betweenall nucleic acids that are 85% identical to one another; hybridizationalso depends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (e.g., the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (e.g., DNA or RNA) affecthybridization. An additional consideration is whether one of the nucleicacids is immobilized (e.g., on a filter).

[0047] An example of a progression from lower to higher stringencyconditions is the following, where the salt content is given as therelative abundance of SSC (a salt solution containing sodium chlorideand sodium citrate; 2X SSC is 10-fold more concentrated than 0.2X SSC).Nucleic acid molecules are hybridized at 42° C. in 2X SSC/0.1% SDS(sodium dodecylsulfate; a detergent) and then washed in 0.2X SSC/0.1%SDS at room temperature (for conditions of low stringency); 0.2XSSC/0.1% SDS at 42° C. (for conditions of moderate stringency); and 0.1XSSC at 68° C. (for conditions of high stringency). Washing can becarried out using only one of the conditions given, or each of theconditions can be used (for example, washing for 10-15 minutes each inthe order listed above). Any or all of the washes can be repeated. Asmentioned above, optimal conditions will vary and can be determinedempirically.

[0048] A second set of conditions that are considered “stringentconditions” are those in which hybridization is carried out at 50° C. inChurch buffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% BSA) and washing iscarried out at 50° C. in 2X SSC.

[0049] Preferably, nucleic acid molecules of the invention that aredefined by their ability to hybridize with nucleic acid molecules havingthe sequence shown in SEQ ID NO:1 under stringent conditions will haveadditional features in common with GLUTX. For example, the nucleic acidmolecules identified by hybridization may have a similar, or identical,expression profile as the GLUTX molecule described herein, or may encodea polypeptide having one or more of the biological activities possessedby GLUTX.

[0050] Once detected, the nucleic acid molecules can be isolated by anyof a number of standard techniques (see, e.g., Sambrook et al.,“Molecular Cloning, A Laboratory Manual,” 2nd Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

[0051] The invention also encompasses: (a) expression vectors thatcontain any of the foregoing GLUTX-related coding sequences and/or theircomplements (i.e. , “antisense” sequence) and fragments thereof; (b)expression vectors that contain any of the foregoing GLUTX-relatedsequences operatively associated with a regulatory element (examples ofwhich are given below) that directs the expression of the codingsequences; (c) expression vectors containing, in addition to sequencesencoding a GLUTX polypeptide, nucleic acid sequences that are unrelatedto nucleic acid sequences encoding GLUTX, such as molecules encoding areporter or marker; and (d) genetically engineered host cells thatcontain any of the foregoing expression vectors, and thereby express thenucleic acid molecules of the invention in the host cell. The regulatoryelements referred to above include, but are not limited to, inducibleand non-inducible promoters, enhancers, operators and other elements,which are known to those skilled in the art, and which drive orotherwise regulate gene expression. Such regulatory elements include butare not limited to the cytomegalovirus hCMV immediate early gene, theearly or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage λ, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

[0052] Additionally, the GLUTX encoding nucleic acid molecules of thepresent invention can form part of a hybrid gene encoding additionalpolypeptide sequences, for example, sequences that function as a markeror reporter. Examples of marker or reporter genes include β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G48^(r)), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacZ (encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, a chimeric or hybrid polypeptide of the inventionwill include a first portion and a second portion; the first portionbeing a GLUTX polypeptide or a fragment thereof (preferably abiologically active fragment) and the second portion being, for example,the reporter described above or an immunoglobulin constant region.

[0053] The expression systems that can be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (e.g.,Saccharomyces and Pichia) transformed with recombinant yeast expressionvectors containing the nucleic acid molecules of the invention(preferably containing a nucleic acid sequence encoding all or a portionof GLUTX (such as the sequence of SEQ ID NO:1); insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing a nucleic acid molecule of the invention; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingGLUTX nucleotide sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, VERO, HeLa, MDCK, W138, and NIH 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., the metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter and the vacciniavirus 7.5 K promoter).

[0054] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, e.g., for the generation of pharmaceuticalcompositions containing GLUTX polypeptides or for raising antibodies tothose polypeptides, vectors that are capable of directing the expressionof high levels of fusion protein products that are readily purified maybe desirable. Such vectors include, but are not limited to, the E. coliexpression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983), in whichthe coding sequence of the insert may be ligated individually into thevector in frame with the lacZ coding region so that a fusion protein isproduced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13:3101-3109, 1985; Van Heeke and Schuster, J. Biol. Chem.264:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0055] In an insect system, Autographa californica nuclear polyhidrosisvirus (AcNPV) can be used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The coding sequence of theinsert may be cloned individually into non-essential regions (e.g., thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (e.g., the polyhedrin promoter). Successful insertion of thecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (e.g., see Smith et al., J. Virol. 46:584,1983; and Smith, U.S. Pat. No. 4,215,051).

[0056] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the nucleic acid molecule of the invention can beligated to an adenovirus transcription/translation control complex, forexample, the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a GLUTX gene product in infectedhosts (e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA81:3655-3659, 1984). Specific initiation signals may also be requiredfor efficient translation of inserted nucleic acid molecules. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where a complete gene or cDNA, including its own initiation codonand adjacent sequences, is inserted into the appropriate expressionvector, no additional translational control signals may be needed.However, in cases where only a portion of the coding sequence isinserted (e.g., the portion encoding the mature form of a GLUTX protein)translational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., Methods in Enzymol.153:516-544, 1987).

[0057] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. The mammalian celltypes listed above are among those that could serve as suitable hostcells.

[0058] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress GLUTX can be engineered. Rather than using expression vectorsthat contain viral origins of replication, host cells can be transformedwith DNA controlled by appropriate expression control elements (e.g.,promoter sequences, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be allowed to growfor 1-2 days in an enriched media, and then switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection, and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which, in turn, canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines that express GLUTX. Such engineered celllines may be particularly useful in screening and evaluating compoundsthat affect the endogenous activity of the gene product (i.e., GLUTX).

[0059] A number of selection systems can be used. For example, theherpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk^(—), hgprt^(—) or aprt^(—) cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA78:2072, 1981); neo, which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147,1984).

[0060] Alternatively, any GLUTX-containing fusion proteins can bereadily purified utilizing an antibody specific for the fusion proteinbeing expressed. For example, a system described by Janknecht et al.allows for the ready purification of non-denatured fusion proteinsexpressed in human cell lines (Proc. Natl. Acad. Sci. USA 88:8972-8976,1991). In this system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the gene's open reading frame istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0061] As implied by the descriptions above, a host cell is any cellinto which (or into an ancestor of which) a nucleic acid encoding apolypeptide of the invention (e.g., a GLUTX polypeptide) has beenintroduced by means of recombinant DNA techniques.

[0062] II. GLUTX Polypeptides

[0063] The GLUTX polypeptides described herein are those encoded by anyof the nucleic acid molecules described above, and include fragments ofGLUTX, mutant forms of GLUTX, active and non-active allelic variants ofGLUTX, splice variants of GLUTX, truncated forms of GLUTX, and fusionproteins containing all or a portion of GLUTX. These polypeptides can beprepared for a variety of uses including, but not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products or exogenous compoundsthat can modulate the activity or expression of GLUTX, and aspharmaceutical reagents useful for the treatment of any disorder inwhich the associated symptoms are improved by altering the activity ofGLUTX.

[0064] The terms “protein” and “polypeptide” are used herein to describeany chain of amino acid residues, regardless of length orpost-translational modification (e.g., modification by glycosylation orphosphorylation). Thus, the term “GLUTX polypeptide” includesfull-length, naturally occurring GLUTX polypeptides (that can bepurified from tissues in which they are naturally expressed, accordingto standard biochemical methods of purification), as well asrecombinantly or synthetically produced polypeptides that correspondeither to a full-length, naturally-occurring GLUTX polypeptide or toparticular domains or portions of such a polypeptide. The term alsoencompasses mature GLUTX having an added amino-terminal methionine(useful for expression in prokaryotic cells).

[0065] Preferred polypeptides are substantially pure GLUTX polypeptidesthat are at least 50% (e.g., 55%, 60%, or 65%), more preferably at least70% (e.g., 72%, 75%, or 78%), even more preferably at least 80% (e.g.,80%, 85% or 90%), and most preferably at least 95% (e.g., 97% or even99%) identical to the sequences encoded by SEQ ID NO:1 (e.g., SEQ IDNO:2). Those of ordinary skill in the art are well able to determine thepercent identity between two amino acid sequences. Thus, if apolypeptide is encoded by a nucleic acid that hybridizes under stringentconditions with the GLUTX CDNA sequence disclosed herein and alsoencodes one or more of the conserved regions present in GLUTX, it willbe recognized as a GLUTX polypeptide and thereby considered within thescope of the present invention.

[0066] The invention also encompasses polypeptides that are functionallyequivalent to GLUTX. These polypeptides are equivalent to GLUTX in thatthey are capable of carrying out one or more of the functions of GLUTXin a biological system. Polypeptides that are functionally equivalent toGLUTX can have 20%, 40%, 50%, 75%, 80%, or even 90% of one or more ofthe biological activities of the full-length, mature human form ofGLUTX. Such comparisons are generally based on an assay of biologicalactivity in which equal concentrations of the polypeptides are used andcompared. The comparison can also be based on the amount of thepolypeptide required to reach 50% of the maximal biological activityobtainable.

[0067] Functionally equivalent proteins can be those, for example, thatcontain additional or substituted amino acid residues. Substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. Amino acids that are typically considered to providea conservative substitution for one another are specified in the Summaryof the Invention.

[0068] Polypeptides that are functionally equivalent to GLUTX can bemade using random mutagenesis techniques well known to those of ordinaryskill in the art (and the resulting mutant GLUTX polypeptides can betested for activity). It is more likely, however, that such polypeptideswill be generated by site-directed mutagenesis (again using techniqueswell known to persons of ordinary skill in the art). These polypeptidesmay have increased functionality or decreased functionality.

[0069] To design functionally equivalent polypeptides, it is useful todistinguish between conserved positions and variable positions. This canbe done by aligning the amino acid sequences of GLUTX that are obtainedfrom various organisms or by aligning GLUTX with other identifiedglucose transporters, e.g., GLUT1 (SEQ ID NO:3), GLUT2 (SEQ ID NO:4),GLUT3 (SEQ ID NO:5), GLUT4 (SEQ ID NO:6), and GLUT5 (SEQ ID NO:7), shownin FIG. 3). Skilled artisans will recognize that conserved amino acidresidues are more likely to be necessary for preservation of function.Thus, it is preferable that conserved residues are not altered.Alignment of GLUTX with other glucose receptors will reveal regions thatare more highly conserved. Such regions are preferably not altered.

[0070] Mutations within the GLUTX coding sequence can be made togenerate variant GLUTX genes that are better suited for expression in aselected host cell. For example, N-linked glycosylation sites can bealtered or eliminated to achieve, for example, expression of ahomogeneous product that is more easily recovered and purified fromyeast hosts which are known to hyperglycosylate N-linked sites. To thisend, a variety of amino acid substitutions at one or both of the firstor third amino acid positions of any one or more of the glycosylationrecognition sequences which occur (in N-X-S or N-X--), and/or an aminoacid deletion at the second position of any one or more of suchrecognition sequences, will prevent glycosylation at the modifiedtripeptide sequence (see, e.g., Miyajima et al., EMBO J. 5:1193, 1986).

[0071] The polypeptides of the invention can be expressed fused toanother polypeptide, for example, a marker polypeptide or fusionpartner. For example, the polypeptide can be fused to a hexa-histidinetag to facilitate purification of bacterially expressed protein or ahemagglutinin tag to facilitate purification of protein expressed ineukaryotic cells. In addition, a GLUTX polypeptide can be fused to GST.

[0072] The polypeptides of the invention can be chemically synthesized(e.g., see Creighton, “Proteins: Structures and Molecular Principles,”W. H. Freeman & Co., NY, 1983), or, perhaps more advantageously,produced by recombinant DNA technology as described herein. Foradditional guidance, persons of ordinary skill in the art may consultAusubel et al. (supra), Sambrook et al. (“Molecular Cloning, ALaboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1989), and, particularly for examples of chemical synthesis, Gait(“Oligonucleotide Synthesis,” IRL Press, Oxford, 1984).

[0073] III. Transgenic animals

[0074] GLUTX polypeptides can also be expressed in transgenic animals.Such transgenic animals represent model systems for the study ofdisorders that are either caused by or exacerbated by misexpression ofGLUTX, or disorders that can be treated by altering the expression ofGLUTX or the activity of GLUTX (even though the expression or activityis not detectably abnormal). Transgenic animals can also be used for thedevelopment of therapeutic agents that modulate the expression of GLUTXor the activity of GLUTX.

[0075] Transgenic animals can be farm animals (e.g., pigs, goats, sheep,cows, horses, rabbits, and the like) rodents (such as rats, guinea pigs,and mice), non-human primates (e.g., baboons, monkeys, and chimpanzees),and domestic animals (e.g., dogs and cats). Transgenic mice areespecially preferred.

[0076] Any technique known in the art can be used to introduce a GLUTXtransgene into animals to produce founder lines of transgenic animals.Such techniques include, but are not limited to, pronuclearmicroinjection (U.S. Pat. No. 4,873,191); retrovirus mediated genetransfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci.,USA 82:6148, 1985); gene targeting into embryonic stem cells (Thompsonet al., Cell 56:313, 1989); and electroporation of embryos (Lo, Mol.Cell. Biol. 3:1803, 1983).

[0077] The present invention provides for transgenic animals that carrya GLUTX transgene in all of their cells, as well as animals that carry atransgene in some, but not all of their cells. For example, theinvention provides for mosaic animals. The GLUTX transgene can beintegrated as a single transgene or in concatamers, for example,head-to-head tandems or head-to-tail tandems. The transgene can also beselectively introduced into, and activated in, a particular cell type(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232, 1992). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art.

[0078] When it is desired that a GLUTX transgene be integrated into thechromosomal site of an endogenous GLUTX gene, gene targeting ispreferred. Briefly, when such a technique is to be used, vectorscontaining some nucleotide sequences homologous to an endogenous GLUTXgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene also can be selectively introduced into a particular celltype, thus inactivating the endogenous GLUTX gene in only that cell type(Gu et al., Science 265:103, 1984). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art. These techniques are useful for preparing “knock outs”having no functional GLUTX gene.

[0079] Once transgenic animals have been generated, the expression ofthe recombinant GLUTX gene can be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to determine whether integration of the transgene has takenplace. The level of mRNA expression of the transgene in the tissues ofthe transgenic animals may also be assessed using techniques whichinclude, but are not limited to, Northern blot analysis of tissuesamples obtained from the animal, in situ hybridization analysis, andRT-PCR. Samples of GLUTX gene-expressing tissue can also be evaluatedimmunocytochemically using antibodies specific for the GLUTX transgeneproduct.

[0080] For a review of techniques that can be used to generate andassess transgenic animals, those of ordinary skill in the art canconsult Gordon (Intl. Rev. Cytol. 115:171-229, 1989), and may obtainadditional guidance from, for example: Hogan et al. “Manipulating theMouse Embryo” (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1986); Krimpenfort et al., Bio/Technology 9:86, 1991; Palmiter et al.,Cell 41:343, 1985; Kraemer et al., “Genetic Manipulation of the EarlyMammalian Embryo,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1985; Hammer et al., Nature 315:680, 1985; Purcel et al., Science244:1281, 1986; Wagner et al., U.S. Pat. No. 5,175,385; and Krimpenfortet al., U.S. Pat. No. 5,175,384.

[0081] The transgenic animals of the invention can be used to determinethe consequence of altering the expression of GLUTX in the context ofvarious disease states. For example, GLUTX knock out mice can begenerated using an established line of mice that serve as a model for adisease in which activity of the missing gene is impaired.

[0082] IV. Anti-GLUTX Antibodies

[0083] GLUTX polypeptides (or immunogenic fragments or analogs thereof)can be used to raise antibodies useful in the invention; suchpolypeptides can be produced by recombinant techniques or synthesized(see, for example, “Solid Phase Peptide Synthesis,” supra; Ausubel etal., supra). In general, GLUTX polypeptides can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Antibodies produced inthat animal can then be purified by peptide antigen affinitychromatography.

[0084] In particular, various host animals can be immunized by injectionwith a GLUTX polypeptide or an antigenic fragment thereof. Commonlyemployed host animals include rabbits, mice, guinea pigs, and rats.Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Potentially useful human adjuvants include BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies areheterogeneous populations of antibody molecules that are contained inthe sera of the immunized animals.

[0085] Antibodies within the invention therefore include polyclonalantibodies and, in addition, monoclonal antibodies, humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, and molecules produced using a Fab expression library.

[0086] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using the GLUTXpolypeptides described above and standard hybridoma technology (see, forexample, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J.Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976;Hammerling et al., “Monoclonal Antibodies and T Cell Hybridomas,”Elsevier, N.Y., 1981; Ausubel et al., supra).

[0087] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.,Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole etal., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this a particularly useful method of production.

[0088] Once produced, polyclonal or monoclonal antibodies are tested forspecific GLUTX recognition by Western blot or immunoprecipitationanalysis by standard methods, for example, as described in Ausubel etal., supra. Antibodies that specifically recognize and bind to GLUTX areuseful in the invention. For example, such antibodies can be used in animmunoassay to monitor the level of GLUTX produced by a mammal (e.g., todetermine the amount or subcellular location of GLUTX).

[0089] Preferably, GLUTX selective antibodies of the invention areproduced using fragments of the GLUTX polypeptide that lie outsidehighly conserved regions and appear likely to be antigenic, by criteriasuch as high frequency of charged residues. FIG. 4 includes a graph ofthe antigenicity index (Jameson-Wolf) for GLUTX. This information can beused to design antigenic peptides. Cross-reactive anti-GLUTX antibodiesare produced using a fragment of GLUTX that is conserved amongst membersof this family of proteins. In one specific example, such fragments aregenerated by standard techniques of PCR, and are then cloned into thepGEX expression vector (Ausubel et al., supra) . Fusion proteins areexpressed in E. coli and purified using a glutathione agarose affinitymatrix as described in Ausubel, et al., supra.

[0090] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusions can be generated for each protein,and each fusion can be injected into at least two rabbits. Antisera canbe raised by injections in a series, preferably including at least threebooster injections.

[0091] Antiserum is also checked for its ability to immunoprecipitaterecombinant GLUTX polypeptides or control proteins, such asglucocorticoid receptor, CAT, or luciferase.

[0092] The antibodies can be used, for example, in the detection ofGLUTX in a biological sample as part of a diagnostic assay or to reduceGLUTX activity as part of a therapeutic regime (e.g., to reduce anundesirable level of GLUTX activity). Antibodies also can be used in ascreening assay to measure the effect of a candidate compound onexpression or localization of GLUTX. Additionally, such antibodies canbe used in conjunction with the gene therapy techniques. For example,they may be used to evaluate the normal and/or engineeredGLUTX-expressing cells prior to their introduction into the patient.

[0093] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1984;Neuberger et al., Nature 312:604, 1984; Takeda et al., Nature 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.

[0094] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692)can be adapted to produce single chain antibodies against a GLUTXpolypeptide, or a fragment thereof. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0095] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0096] Antibodies can be humanized by methods known in the art. Forexample, monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and5,569,825).

[0097] The methods described herein, in which anti-GLUTX antibodies areemployed, can be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one specific antibody reagentdescribed herein, which may be conveniently used, for example, inclinical settings, to diagnose patients exhibiting symptoms of thedisorders associated with aberrant expression of GLUTX.

[0098] V. Antisense Nucleic Acid Molecules

[0099] Treatment regimes based on an “antisense” approach involve thedesign of oligonucleotides (either DNA or RNA) that are complementary toa portion of a selected mRNA. These oligonucleotides bind tocomplementary mRNA transcripts and prevent their translation. Absolutecomplementarity, although preferred, is not required. A sequence“complementary” to a portion of an RNA molecule, as referred to herein,is a sequence having sufficient complementarily to hybridize with theRNA, forming a stable duplex; in the case of double-stranded antisensenucleic acids, a single strand of the duplex DNA can be tested, ortriplex formation can be assayed. The ability to hybridize will dependon both the degree of complementarily and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with an RNA it may contain and still form a stableduplex (or triplex, as the case may be). One of ordinary skill in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0100] Oligonucleotides that are complementary to the 5′ end of themessage, for example, the 5′ untranslated sequence up to and includingthe AUG initiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs recently have been shown to be effective atinhibiting translation of mRNAs as well (Wagner, Nature 372:333, 1984).Thus, oligonucleotides complementary to either the 5′ or 3′non-translated, non-coding regions of a GLUTX gene, could be used in anantisense approach to inhibit translation of endogenous GLUTX- mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon.

[0101] Antisense oligonucleotides complementary to mRNA coding regionsare less efficient inhibitors of translation but could be used inaccordance with the invention. Whether designed to hybridize to the 5′,3′, or coding region of GLUTX mRNA, antisense nucleic acids should be atleast six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspects,the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides, or at least 50 nucleotides.

[0102] Regardless of the choice of target sequence, as with othertherapeutic strategies directed to GLUTX, it is preferred that in vitrostudies are first performed to assess the ability of an antisenseoligonucleotide to inhibit gene expression. If desired, the assessmentcan be quantitative. It is preferred that these studies utilize controlsthat distinguish between antisense gene inhibition and any nonspecificbiological effect that an oligonucleotide may cause. It is alsopreferred that these studies compare levels of the target RNA or proteinwith that of an internal control RNA or protein. Additionally, it isenvisioned that results obtained using an antisense oligonucleotide arecompared with those obtained using a control oligonucleotide.Preferably, the control oligonucleotide is of approximately the samelength as the test oligonucleotide, and the nucleotide sequence of thecontrol oligonucleotide differs from that of the test antisense sequenceno more than is necessary to prevent specific hybridization between thecontrol oligonucleotide and the targeted RNA sequence.

[0103] The oligonucleotides can contain DNA or RNA, or they can containchimeric mixtures, derivatives, or modified versions thereof that areeither single-stranded or double-stranded. The oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone, forexample, to improve stability of the molecule, hybridization, etc.Modified sugar moieties can be selected from the group including, butnot limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. Amodified phosphate backbone can be selected from the group consisting ofa phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or an analog of any of thesebackbones.

[0104] The oligonucleotide can include other appended groups such aspeptides (e.g., for disrupting the transport properties of the moleculein host cells in vivo), or agents that facilitate transport across thecell membrane (as described, for example, in Letsinger et al., Proc.Natl. Acad. Sci. USA 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad.Sci. USA 84:648, 1987; PCT Publication No. WO 88/09810) or theblood-brain barrier (see, for example, PCT Publication No. WO 89/10134),or hybridization-triggered cleavage agents (see, for example, Krol etal., BioTechniques 6:958, 1988), or intercalating agents (see, forexample, Zon, Pharm. Res. 5:539, 1988). To this end, the oligonucleotidecan be conjugated to another molecule, for example, a peptide, ahybridization triggered cross-linking agent, a transport agent, or ahybridization-triggered cleavage agent.

[0105] An antisense oligonucleotide of the invention can comprise atleast one modified base moiety that is selected from the groupincluding, but not limited to, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

[0106] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids. Res. 15:6625, 1987). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett. 215:327,1987).

[0107] Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, for example, by use of an automatedDNA synthesizer (such as are commercially available from Biosearch,Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides can be synthesized by the method of Stein et al. (Nucl.Acids Res. 16:3209, 1988), and methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports (Sarin et al.,Proc. Natl. Acad. Sci. USA 85:7448, 1988).

[0108] For therapeutic application, antisense molecules of the inventionshould be delivered to cells that express GLUTX in vivo. A number ofmethods have been developed for delivering antisense DNA or RNA tocells; for example, antisense molecules can be injected directly intothe tissue site. Alternatively, modified antisense molecules, which aredesigned to target cells that express GLUTX (e.g., antisense moleculeslinked to peptides or antibodies that specifically bind receptors orantigens expressed on the target cell surface) can be administeredsystemically.

[0109] However, it is often difficult to achieve intracellularconcentrations of antisense molecules that are sufficient to suppresstranslation of endogenous mRNAs. Therefore, a preferred approach uses arecombinant DNA construct in which the antisense oligonucleotide isplaced under the control of a strong pol III or pol II promoter. The useof such a construct to transfect target cells in the patient will resultin the transcription of sufficient amounts of single stranded RNAs thatwill form complementary base pairs with endogenous GLUTX transcripts andthereby prevent translation of GLUTX mRNA. For example, a vector can beintroduced in vivo in such a way that it is taken up by a cell andthereafter directs the transcription of an antisense RNA. Such a vectorcan remain episomal or become chromosomally integrated, as long as itcan be transcribed to produce the desired antisense RNA.

[0110] Vectors encoding a GLUTX antisense sequence can be constructed byrecombinant DNA technology methods that are standard practice in theart. Suitable vectors include plasmid vectors, viral vectors, or othertypes of vectors known or newly discovered in the art. The criterion foruse is only that the vector be capable of replicating and expressing theGLUTX antisense molecule in mammalian cells. Expression of the sequenceencoding the antisense RNA can be directed by any promoter known in theart to act in mammalian, and preferably in human, cells. Such promoterscan be inducible or constitutively active and include, but are notlimited to: the SV40 early promoter region (Bernoist et al., Nature290:304, 1981); the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., Cell 22:787-797, 1988); the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA78:1441, 1981); or the regulatory sequences of the metallothionein gene(Brinster et al., Nature 296:39, 1988).

[0111] VI. Ribozymes

[0112] Ribozyme molecules designed to catalytically cleave GLUTX mRNAtranscripts also can be used to prevent translation of GLUTX mRNA andexpression of GLUTX polypeptides (see, for example, PCT Publication WO90/11364; Saraver et al., Science 247:1222, 1990). While variousribozymes that cleave mRNA at site-specific recognition sequences can beused to destroy GLUTX mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art (Haseloff et al., Nature334:585, 1988). There are numerous examples of potential hammerheadribozyme cleavage sites within the nucleotide sequence of human GLUTXcDNA. Preferably, the ribozyme is engineered so that the cleavagerecognition site is located near the 5′ end of the GLUTX mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

[0113] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena Thermophila (known as the IVS orL-19 IVS RNA), and which has been extensively described by Cech and hiscollaborators (Zaug et al., Science 224:574, 1984; Zaug et al., Science231:470, 1986; Zug et al., Nature 324:429, 1986; PCT Application No. WO88/04300; and Been et al., Cell 47:207, 1986). The Cech-type ribozymeshave an eight base-pair sequence that hybridizes to a target RNAsequence, whereafter cleavage of the target RNA takes place. Theinvention encompasses those Cech-type ribozymes that target eightbase-pair active site sequences present in GLUTX.

[0114] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.), and should be delivered to cells which express the GLUTX in vivo.A preferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous GLUTX messages andinhibit translation. Because ribozymes, unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[0115] VII. Peptide Nucleic Acids

[0116] Nucleic acid molecules encoding GLUTX (or a fragment thereof) canbe modified at the base moiety, sugar moiety, or phosphate backbone toimprove, for example, the stability or solubility of the molecule or itsability to hybridize with other nucleic acid molecules. For example, thedeoxyribose phosphate backbone of the nucleic acid can be modified togenerate peptide nucleic acids (see Hyrup et al., Bioorganic Med. Chem.4:5-23 (1996). As used herein, the terms “peptide nucleic acids” or“PNAs” refer to nucleic acid mimics, for example, DNA mimics, in whichthe deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al., supra; Perry-O'Keefe et al.Proc. Natl. Acad. Sci. USA 93:14670-14675 (1996).

[0117] PNAs of GLUTX can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, forexample, inducing transcription or translation arrest or inhibitingreplication. PNAs of GLUTX can also be used, for example, in theanalysis of single base pair mutations in a gene by, for example,PNA-directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, for example, S1 nucleases (Hyrup etal., supra); or as probes or primers for DNA sequence and hybridization(Hyrup et al., supra; Perry-O'Keefe, supra).

[0118] In other embodiments, PNAs of GLUTX can be modified, for example,to enhance their stability or cellular uptake, by attaching lipophilicor other helper groups to the PNA, by the formation of PNA-DNA chimeras,or by the use of liposomes or other techniques of drug delivery known inthe art. For example, PNA-DNA chimeras of GLUTX can be generated thatmay combine the advantageous properties of PNA and DNA. Such chimerasallow DNA recognition enzymes, for example, RNAse H and DNA polymerases,to interact with the DNA portion while the PNA portion would providehigh binding affinity and specificity. PNA-DNA chimeras can be linkedusing linkers of appropriate lengths selected in terms of base stacking,number of bonds between the nucleobases, and orientation (Hyrup et al.,supra). The synthesis of PNA-DNA chimeras can be performed as describedin Hyrup, supra, and Finn et al., Nucl. Acids Res. 24:3357-3363 (1996).For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al., Nucl. Acids Res. 17:5973-5988, 1989). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al., supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser et al., Bioorganic Med. Chem. Lett. 5:1119-11124(1975).

[0119] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA84:648-652 (1987); PCT Publication No. WO 88/09810, published Dec. 15,1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., BioTech. 6:958-976 (1988)) or integrating agents (see, e.g.,Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleotide maybe conjugated to another molecule, for example, a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent etc.

[0120] VIII. Proteins that Associate with GLUTX

[0121] The invention also features methods for identifying polypeptidesthat can associate with GLUTX, as well as the isolated interactingprotein. Any method that is suitable for detecting protein-proteininteractions can be employed to detect polypeptides that associate withGLUTX, whether these polypeptides associate with the transmembrane,intracellular, or extracellular domains of GLUTX. Among the traditionalmethods that can be employed are co-immuno-precipitation, crosslinking,and co-purification through gradients or chromatographic columns of celllysates or proteins obtained from cell lysates and the use of GLUTX toidentify proteins in the lysate that interact with GLUTX. For theseassays, the GLUTX polypeptide can be a full length GLUTX, anextracellular domain of GLUTX, or some other suitable GLUTX polypeptide.Once isolated, such an interacting protein can be identified and clonedand then used, in conjunction with standard techniques, to alter theactivity of the GLUTX polypeptide with which it interacts. For example,at least a portion of the amino acid sequence of a protein thatinteracts with GLUTX can be ascertained using techniques well known tothose of skill in the art, such as via the Edman degradation technique.The amino acid sequence obtained can be used as a guide for thegeneration of oligonucleotide mixtures that can be used to screen forgene sequences encoding the interacting protein. Screening can beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and thescreening are well-known (Ausubel, supra; and “PCR Protocols: A Guide toMethods and Applications,” Innis et al., eds. Academic Press, Inc., NY,1990).

[0122] Additionally, methods can be employed that result directly in theidentification of genes that encode proteins that interact with GLUTX.These methods include, for example, screening expression libraries, in amanner similar to the well known technique of antibody probing of λgt11libraries, using labeled GLUTX polypeptide or a GLUTX fusion protein,for example, a GLUTX polypeptide or domain fused to a marker such as anenzyme, fluorescent dye, a luminescent protein, or to an IgFc domain.

[0123] There are also methods available that can detect protein-proteininteraction in vivo. A method which detects protein interactions in vivois the two-hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA88:9578, 1991). A kit for practicing this method is available fromClontech (Palo Alto, Calif.).

[0124] Briefly, utilizing such a system, plasmids are constructed thatencode two hybrid proteins: one plasmid includes a nucleotide sequenceencoding the DNA-binding domain of a transcription activator proteinfused to a nucleotide sequence encoding GLUTX, a GLUTX polypeptide, or aGLUTX fusion protein, and the other plasmid includes a nucleotidesequence encoding the transcription activator protein's activationdomain fused to a cDNA encoding an unknown protein which has beenrecombined into this plasmid as part of a cDNA library. The DNA-bindingdomain fusion plasmid and the cDNA library are transformed into a strainof the yeast Saccharomyces cerevisiae that contains a reporter gene(e.g., HBS or LacZ) whose regulatory region contains the transcriptionactivator's binding site. Either hybrid protein alone cannot activatetranscription of the reporter gene: the DNA-binding domain hybrid cannotbecause it does not provide activation function, and the activationdomain hybrid cannot because it cannot localize to the activator'sbinding sites. Interaction of the two hybrid proteins reconstitutes thefunctional activator protein and results in expression of the reportergene, which is detected by an assay for the reporter gene product.

[0125] The two-hybrid system or related methodology can be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation,GLUTX may be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of bait GLUTX gene product fusedto the DNA-binding domain are co-transformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, a bait GLUTX gene sequence, suchas that encoding GLUTX or a domain of GLUTX can be cloned into a vectorsuch that it is translationally fused to the DNA encoding theDNA-binding domain of the GAL4 protein. These colonies are purified andthe library plasmids responsible for reporter gene expression areisolated. DNA sequencing is then used to identify the proteins encodedby the library plasmids.

[0126] A cDNA library of the cell line from which proteins that interactwith bait GLUTX gene product are to be detected can be made usingmethods routinely practiced in the art. According to the particularsystem described herein, for example, the cDNA fragments can be insertedinto a vector such that they are translationally fused to thetranscriptional activation domain of GAL4. This library can beco-transformed along with the bait GLUTX gene-GAL4 fusion plasmid into ayeast strain which contains a lacZ gene driven by a promoter whichcontains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with bait GLUTX geneproduct will reconstitute an active GAL4 protein and thereby driveexpression of the HIS3 gene. Colonies that express HIS3 can then bepurified from these strains and used to produce and isolate the baitGLUTX gene-interacting protein using techniques routinely practiced inthe art.

[0127] IX. Detection of GLUTX or Nucleic Acid Molecules Encoding GLUTXand Related Diagnostic Assays

[0128] The invention encompasses methods for detecting the presence ofGLUTX protein or nucleic acid in a biological sample as well as methodsfor measuring the level of GLUTX protein or nucleic acid in a biologicalsample. Such methods are useful for diagnosis of disorders associatedwith aberrant expression of GLUTX.

[0129] An exemplary method for detecting the presence or absence ofGLUTX in a biological sample involves obtaining a biological sample froma test subject and contacting the biological sample with a compound oran agent capable of detecting a GLUTX polypeptide or a GLUTX nucleicacid (e.g., mRNA or genomic DNA). A preferred agent for detecting GLUTXmRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to GLUTX mRNA or genomic DNA. The nucleic acid probe can be,for example, a full-length GLUTX nucleic acid molecule, such as anucleic acid molecule having the sequence of SEQ ID NO:l, or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250, or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to GLUTX mRNA or genomic DNA.

[0130] A preferred agent for detecting a GLUTX polypeptide is anantibody capable of binding to an GLUTX polypeptide, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used. The term “labeled,” with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin. The term “biological sample” isintended to include tissues, cells, and biological fluids isolated froma subject, as well as tissues, cells and fluids present within asubject. That is, the detection method of the invention can be used todetect GLUTX mRNA, a GLUTX polypeptide, or GLUTX genomic DNA in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of GLUTX mRNA include Northern hybridizationsand in situ hybridizations. In vitro techniques for detection of a GLUTXpolypeptide include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of GLUTX genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a GLUTX polypeptideinclude introducing into a subject a labeled anti-GLUTX antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

[0131] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject.

[0132] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting a GLUTXpolypeptide, GLUTX mRNA, or GLUTX genomic DNA, such that the presence ofa GLUTX polypeptide, GLUTX mRNA, or GLUTX genomic DNA is detected in thebiological sample, and comparing the presence of GLUTX polypeptide,GLUTX mRNA, or genomic DNA in the control sample with the presence ofGLUTX polypeptides, mRNA or genomic DNA in a test sample.

[0133] The invention also encompasses kits for detecting the presence ofGLUTX nucleic acid molecules or GLUTX polypeptides in a biologicalsample. For example, the kit can contain a labeled compound or agentcapable of detecting a GLUTX polypeptide or a GLUTX mRNA molecule in abiological sample; means for determining the amount of GLUTX in thesample; and means for comparing the amount of GLUTX in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further contain instructions for using the kit to detect aGLUTX polypeptide or GLUTX nucleic acid molecule.

[0134] X. Prognostic Assays

[0135] The invention also encompasses prognostic assays that can be usedto identify subjects having or at risk of developing a disease ordisorder associated with aberrant GLUTX expression or GLUTX activity.Thus, the present invention provides methods in which a test sample isobtained from a subject and the level, or presence, or allelic formGLUTX nucleic acid molecules or GLUTX polypeptides ia assessed. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), a cell sample, or tissue.

[0136] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, nucleic acid, smallmolecule or other drug candidate) to treat a disease or disorderassociated with aberrant GLUTX expression or GLUTX activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent that modulates GLUTX expression and/oractivity. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant GLUTX expression or GLUTX activity inwhich a test sample is obtained and GLUTX nucleic acids or GLUTXpolypeptides are detected (e.g., wherein the presence of a particularlevel of GLUTX expression or a particular GLUTX allelic variant isdiagnostic for a subject that can be administered an agent to treat adisorder associated with aberrant GLUTX expression or GLUTX activity).

[0137] The methods of the invention can also be used to detect geneticalterations in a GLUTX. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one alterationaffecting the integrity of the gene encoding a GLUTX polypeptide or themisexpression of the GLUTX gene. For example, such genetic alterationscan be detected by ascertaining the existence of at least one of: (1) adeletion of one or more nucleotides from a GLUTX gene; (2) an additionof one or more nucleotides to a GLUTX gene; (3) a substitution of one ormore nucleotides of a GLUTX gene; (4) a chromosomal rearrangement of aGLUTX gene; (5) an alteration in the level of a messenger RNA transcriptof a GLUTX gene; (6) aberrant modification of a GLUTX gene, such as ofthe methylation pattern of the genomic DNA, (7) the presence of anon-wild type splicing pattern of a messenger RNA transcript of a GLUTXgene; and (10) inappropriate post-translational modification of a GLUTXpolypeptide. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting alterationsin a GLUTX gene.

[0138] In certain embodiments, detection of the alteration involves theuse of a probe/primer in a polymerase chain reaction (PCR; see, e.g.,U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or alternatively, in a ligation chain reaction (LCR; see, e.g.,Landegran et al., Science 241:1077-1080, 1988; and Nakazawa et al. Proc.Natl. Acad. Sci. USA 91:360-364, 1994), the latter of which can beparticularly useful for detecting point mutations in the GLUTX gene (seeAbavaya et al., Nucl. Acids Res. 23:675-681, 1995). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic DNA, mRNA, or both) from the cellsof the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a GLUTX gene under conditionssuch that hybridization and amplification of the GLUTX nucleic acid (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

[0139] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al., Proc. Natl. Acad. Sci USA87:1874-1878, 1990), transcriptional amplification system (Kwoh et al.,Proc. Natl. Acad. Sci USA 86:1173-1177, 1989), Q-Beta Replicase (Lizardiet al., Bio/Technology 6:1197, 1988), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of ordinary skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very low number.

[0140] In an alternative embodiment, alterations in a GLUTX gene from asample cell can be identified by identifying changes in a restrictionenzyme cleavage pattern. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0141] In other embodiments, alterations in GLUTX can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing tens to thousands of oligonucleotideprobes (Cronin et al., Human Mutation 7:244-255, 1996); Kozal et al.,Nature Medicine 2:753-759, 1996). For example, alterations in GLUTX canbe identified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

[0142] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the GLUTXgene and detect mutations by comparing the sequence of the sample GLUTXwith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert (Proc. Natl. Acad. Sci. USA 74:560 (1977)) or Sanger(Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that anyof a variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Bio/Techniques 19:448, 1995) includingsequencing by mass spectrometry (see, e.g. PCT International PublicationNo. WO 94/16101; Cohen et al. Adv. Chromatogr. 36:127-162 1996; andGriffin et al., Appl. Biochem. Biotechnol. 38:147-159, 1993).

[0143] Other methods of detecting mutations in the GLUTX gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.Science 230:1242 1985). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes formed by hybridizing(labeled) RNA or DNA containing the wild-type GLUTX sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. (see, forexample, Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397 1988; Saleebaet al., Methods Enzymol. 217:286-295 1992). In a preferred embodiment,the control DNA or RNA can be labeled for detection.

[0144] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in GLUTX cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches (Hsu et al., Carcinogenesis 15:1657-16621994). According to an exemplary embodiment, a probe based on a GLUTXsequence is hybridized to a cDNA or other DNA product from a test cellor cells. The duplex is treated with a DNA mismatch repair enzyme, andthe cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0145] In other embodiments, alterations in electrophoretic mobility canbe used to identify mutations in GLUTX genes. For example, single strandconformation polymorphism (SSCP) can be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton MutatRes. 285:125-144 1993; and Hayashi Genet. Anal. Tech. Appl. 9:73-791992). Single-stranded DNA fragments of sample and control GLUTX nucleicacids will be denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Kee et al., Trends Genet. 7:5 1991).

[0146] In yet another embodiment, the movement of mutant or wild-typefragments in a polyacrylamide gel containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE; Myers etal., Nature 313:495, 1985). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denture, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum et al., Biophys. Chem.265:12753, 1987).

[0147] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al., Nature 324;163, 1986); Saiki et al., Proc. NAtl. Acad.Sci. USA 86:6230, 1989). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0148] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule, so that amplification depends on differential hybridization(Gibbs et al., Nucl. Acids Res. 17:2437-2448, 1989) or at the extreme 3′end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner, Tib/Tech 11:238,1993). In addition it may be desirable to introduce a novel restrictionsite in the region of the mutation to create cleavage-based detection(Gasparini et al., Mol. Cell Probes 6:1, 1992). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany, Proc. Natl. Acad. Sci. USA 88:89,1991). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence of absence of amplification.

[0149] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, for example, in a clinical setting to diagnosepatient exhibiting symptoms or a family history of a disease or disorderinvolving abnormal GLUTX activity.

[0150] XI. Pharmacogenetics

[0151] Agents or modulators which have a stimulatory or inhibitoryeffect on GLUTX activity (including those that alter activity byaltering GLUTX gene expression), identified by a screening assaydescribed herein, can be administered to individuals to treat,prophylactically or therapeutically, disorders associated with aberrantGLUTX activity. In conjunction with such treatment, the pharmacogenetics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) of theindividual may be considered. Thus, the pharmacogenetics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenetics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of GLUTX polypeptides, expression of GLUTX nucleic acids, orsequence of GLUTX genes in an individual can be determined and used tothereby select an appropriate agent for therapeutic or prophylactictreatment of the individual.

[0152] Pharmacogenetics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons (See, e.g., Eichelbaum, Clin. Exp.Pharmacol. Physiol. 23:983-985, 1996 and Linder, Clin. Chem. 43:254-266,1997). In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as single factorsaltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0153] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase (NAT2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the excessive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme is the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0154] Thus, the activity of GLUTX polypeptide, expression of GLUTXnucleic acid, or the precise sequence of a GLUTX gene in an individualcan be determined and used to select an appropriate agent fortherapeutic or prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with a GLUTX modulator, such as a modulatoridentified by one of the exemplary screening assays described herein.

[0155] XII. Monitoring of Clinical Trials

[0156] Monitoring the influence of agents (e.g., drugs, compounds) onthe expression of GLUTX or the activity of GLUTX can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase GLUTX gene expression, increase GLUTX polypeptidelevels, or upregulate GLUTX activity, can be monitored in clinicaltrials of subjects exhibiting decreased GLUTX gene expression, decreasedGLUTX polypeptide levels, or downregulated GLUTX activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease GLUTX gene expression, decrease GLUTX polypeptidelevels, or downregulate GLUTX activity, can be monitored in clinicaltrials of subjects exhibiting increased GLUTX gene expression, increasedGLUTX polypeptide levels, or upregulated GLUTX activity. In suchclinical trials, the expression of GLUTX or activity of GLUTX can beused as a measure of the responsiveness of a particular cell.

[0157] For example, and not by way of limitation, genes, includingGLUTX, that are modulated in cells by treatment with an agent (e.g., acompound, drug, or small molecule) that modulates GLUTX activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of agents on a given disorder, for example, ina clinical trial, the level or expression of GLUTX or other genesimplicated in the disorder can be measured. The levels of geneexpression (i.e., a gene expression pattern) can be quantified byNorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of polypeptide produced, by one of the methodsdescribed herein, or by measuring the levels of activity of GLUTX orother genes. In this way, the gene expression pattern can serve as anindicative marker of the physiological response of the cells to theagent. Accordingly, this response state can be determined before, and atvarious points during, treatment of the individual with the agent.

[0158] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide,nucleic acid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (2) detecting the level of expression of a GLUTX polypeptideor GLUTX mRNA in the pre-administration sample, or the level or activityof GLUTX; (3) obtaining one or more post-administration samples from thesubject; (4) detecting the level of expression of GLUTX polypeptide orGLUTX mRNA or the level or activity of the GLUTX polypeptide in thepost-administration sample; (5) comparing the level of expression ofGLUTX mRNA in the pre-administration sample with that in thepost-administration sample, or comparing the level or activity of theGLUTX polypeptide in the pre-administration sample with that in thepost-administration sample; and (6) altering the administration of theagent to the subject accordingly.

[0159] XIII. Screening Assays for Compounds that Modulate GLUTXExpression or Activity

[0160] The invention also encompasses methods for identifying compoundsthat interact with GLUTX (or a domain of GLUTX) including, but notlimited to, compounds that interfere with the interaction of GLUTX withtransmembrane, extracellular, or intracellular proteins which regulateGLUTX activity and compounds which modulate GLUTX activity. Alsoencompasses are method for identifying compounds which bind to GLUTXgene regulatory sequences (e.g., promoter sequences) and which maymodulate GLUTX gene expression.

[0161] The compounds which may be screened in accordance with theinvention include, but are not limited to peptides, antibodies andfragments thereof, and other organic compounds that bind to GLUTX andincrease or decrease activity.

[0162] Such compounds may include, but are not limited to, peptides suchas, for example, soluble peptides, including but not limited to membersof random peptide libraries; (Lam et al., Nature 354:82-84, 1991;Houghten et al., Nature 354:84-86, 1991), and combinatorialchemistry-derived molecular library made of D- and/or L configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries;Songyang, et al., Cell 72:767-778, 1993), antibodies (including, but notlimited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimericor single chain antibodies, and FAb, F(ab′)₂ and FAb expression libraryfragments, and epitope-binding fragments thereof), and small organic orinorganic molecules.

[0163] Other compounds which can be screened in accordance with theinvention include but are not limited to small organic molecules thatare able to gain entry into an appropriate cell and affect theexpression of the GLUTX gene or activity of GLUTX protein.

[0164] Computer modelling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate GLUTX expression or activity. Havingidentified such a compound or composition, the active sites or regionsare identified. Such active sites might typically be a binding for anatural modulator of activity. The active site can be identified usingmethods known in the art including, for example, from the amino acidsequences of peptides, from the nucleotide sequences of nucleic acids,or from study of complexes of the relevant compound or composition withits natural ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the modulator (or ligand) is found.

[0165] Next, the three dimensional geometric structure of the activesite is determined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed modulator (ligand), natural or artificial, which may increasethe accuracy of the active site structure determined.

[0166] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer-based numerical modelling can beused to complete the structure or improve its accuracy. Any recognizedmodelling method may be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0167] Finally, having determined the structure of the active site,either experimentally, by modeling, or by a combination, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. These compounds found from this search are potential GLUTXmodulating compounds.

[0168] Alternatively, these methods can be used to identify improvedmodulating compounds from a previously identified modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modelling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0169] Examples of molecular modelling systems are the CHARMm and QUANTAprograms (Polygen Corporation; Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0170] A number of articles review computer modelling of drugsinteractive with specific proteins, such as Rotivinen et al., ActaPharmaceutical Fennica 97:159-166, 1993; Ripka, New Scientist 54-57(Jun. 16, 1988); McKinaly and Rossmann, Annu. Rev. Pharmacol. Toxiciol.29:111-122, 1989; Perry and Davies, OSAR: QuantitativeStructure-Activity Relationships in Drug Design, pp. 189-193 (Alan R.Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236:125-140and 141-162, 1980; and, with respect to a model receptor for nucleicacid components, Askew et al., J. Am. Chem. Soc. 111:1082, 1989.

[0171] Other computer programs that screen and graphically depictchemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

[0172] Although described above with reference to design and generationof compounds which could alter binding, one could also screen librariesof known compounds, including natural products or synthetic chemicals,and biologically active materials, including proteins, for compoundswhich are inhibitors or activators of GLUTX activity

[0173] Compounds identified via assays such as those described hereinmay be useful, for example, in elaborating the biological function ofGLUTX and for the treatment of disorders associated with aberrant GLUTXactivity or expression. Assays for testing the effectiveness ofcompounds identified with the above-described techniques are discussedbelow.

[0174] In vitro systems may be designed to identify compounds capable ofinteracting with GLUTX (or a domain of GLUTX). Compounds identified maybe useful, for example, in modulating the activity of wild type and/ormutant GLUTX; may be useful in elaborating the biological functionGLUTX; may be utilized in screens for identifying compounds that disruptnormal GLUTX interactions; or may in themselves disrupt suchinteractions.

[0175] The principle of the assays used to identify compounds that bindto GLUTX involves preparing a reaction mixture of GLUTX (or a domainthereof) and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The GLUTX species used can vary depending upon the goal of thescreening assay. In some situations it is preferable to employ a peptidecorresponding to a domain of GLUTX fused to a heterologous protein orpolypeptide that affords advantages in the assay system (e.g., labeling,isolation of the resulting complex, etc.) can be utilized.

[0176] The screening assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay involves anchoring GLUTXprotein, polypeptide, peptide or fusion protein or the test substanceonto a solid phase and detecting GLUTX/test compound complexes anchoredon the solid phase at the end of the reaction. In one embodiment of sucha method, the GLUTX reactant may be anchored onto a solid surface, andthe test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

[0177] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

[0178] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynon-immobilized component (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).

[0179] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for GLUTXprotein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

[0180] Alternatively, cell-based assays can be used to identifycompounds that interact with GLUTX. To this end, cell lines that expressGLUTX, or cell lines that have been genetically engineered to expressGLUTX can be used.

[0181] XIV. Assays for Compounds that Interfere with the InteractionBetween GLUTX and a Protein Binding Partner

[0182] Proteins that interact with the GLUTX are referred to, forpurposes of this discussion, as “binding partners”. Such bindingpartners can be involved in regulating GLUTX activity. Therefore, it isdesirable to identify compounds that interfere with or disrupt theinteraction of such binding partners with GLUTX. Such compounds may beuseful in regulating the activity of the GLUTX and treating disordersassociated with aberrant GLUTX activity.

[0183] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between the GLUTX andbinding partner or partners involves preparing a reaction mixturecontaining GLUTX protein, polypeptide, peptide or fusion protein and thebinding partner under conditions and for a time sufficient to allow thetwo to interact and bind, thus forming a complex. In order to test acompound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of the GLUTX moiety and its binding partner.Control reaction mixtures are incubated without the test compound orwith a non-active control compound. The formation of any complexesbetween the GLUTX moiety and the binding partner is then detected. Theformation of a complex in the control reaction, but not in the reactionmixture containing the test compound, indicates that the compoundinterferes with the interaction of GLUTX and the interactive bindingpartner. Additionally, complex formation within reaction mixturescontaining the test compound and normal GLUTX protein may also becompared to complex formation within reaction mixtures containing thetest compound and a mutant GLUTX. This comparison may be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal GLUTX.

[0184] The assay for compounds that interfere with the interaction ofthe GLUTX and a binding partner can be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either theGLUTX protein, polypeptide, peptide, or fusion protein, or the bindingpartner onto a solid phase and detecting complexes anchored on the solidphase at the end of the reaction. In homogeneous assays, the entirereaction is carried out in a liquid phase. In either approach, the orderof addition of reactants can be varied to obtain different informationabout the compounds being tested. For example, test compounds thatinterfere with the interaction by competition can be identified byconducting the reaction in the presence of the test substance; i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the GLUTX moiety and interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

[0185] In a heterogeneous assay system, either the GLUTX moiety or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of GLUTX (or a domain thereof) or bindingpartner and drying. Alternatively, an immobilized antibody specific forthe species to be anchored may be used to anchor the species to thesolid surface. The surfaces may be prepared in advance and stored.

[0186] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thenon-immobilized species is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe non-immobilized species is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface, e.g., using a directlyor indirectly labeled antibody specific for the initiallynon-immobilized species. Depending upon the order of addition ofreaction components, test compounds which inhibit complex formation orwhich disrupt preformed complexes can be detected.

[0187] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected, e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

[0188] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of the GLUTX moietyand the interactive binding partner is prepared in which either theGLUTX or its binding partners is labeled, but the signal generated bythe label is quenched due to formation of the complex (see, e.g., U.S.Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances which disrupt GLUTX/intracellular binding partner interactioncan be identified.

[0189] In a particular embodiment, a GLUTX fusion can be prepared forimmobilization. For example, the GLUTX or a peptide fragment thereof canbe fused to a glutathione-S-transferase (GST) gene using a fusionvector, such as PGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive bindingpartner can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art. This antibody can be labeledwith the radioactive isotope ¹²⁵I, for example, by methods routinelypracticed in the art. In a heterogeneous assay, the GST-GLUTX fusionprotein can be anchored to glutathione-agarose beads. The interactivebinding partner can then be added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between GLUTX andthe interactive binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

[0190] Alternatively, the GST-GLUTX fusion protein and the interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of inhibition of the GLUTX/binding partnerinteraction can be detected by adding the labeled antibody and measuringthe radioactivity associated with the beads.

[0191] In another embodiment of the invention, these same techniques canbe employed using peptide fragments that correspond to the bindingdomains of GLUTX and/or the interactive or binding partner (in caseswhere the binding partner is a protein), in place of one or both of thefull length proteins. Any number of methods routinely practiced in theart can be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

[0192] XV. Methods for Reducing GLUTX Expression

[0193] Expression of GLUTX can be reduced through the use of modulatorycompounds identified through the use of the screening methods describedabove. In addition, endogenous GLUTX gene expression can also be reducedby inactivating or “knocking out” the GLUTX gene or its promoter usingtargeted homologous recombination (see, for example, U.S. Pat. No.5,464,764). For example, a mutant, non-functional GLUTX (or a completelyunrelated DNA sequence) flanked by DNA omologous to the endogenous GLUTXgene (either the coding egions or regulatory regions of the GLUTX gene)can be sed, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express GLUTX-3 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the GLUTX gene. Such approaches areparticularly suited for use in developing animal models to study therole of GLUTX; in this instance, modifications to ES (embryonic stem)cells can be used to generate animal offspring with an inactive GLUTXgene. However, a knock out approach can be adapted for use in humans,provided the recombinant DNA constructs are directly administered ortargeted to the required site in vivo using appropriate viral vectors.

[0194] Alternatively, endogenous GLUTX gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the GLUTX gene (i.e., the GLUTX promoter and/or enhancers) toform triple helical structures that prevent transcription of the GLUTXgene in target cells in the body (Helene, Anticancer Drug Res. 6:569,1981; Helene et al., Ann. N.Y. Acad. Sci. 660:27, 1992; and Maher,Bioassays 14:807, 1992).

[0195] In addition, as discussed above, anti-sense molecules, ribozymes,and peptide nucleic acids can be used to reduce GLUTX expression.

[0196] VI. Assays for the Identification of Compounds that AmeliorateDisorders Associated with Aberrant GLUTX Expression or Activity

[0197] Compounds, including, but not limited to, compounds identifiedvia assay techniques such as those described above may be useful for thetreatment of disorders associated with aberrant GLUTX expression oraberrant GLUTX activity.

[0198] While animal model-based assays are particularly useful for theidentification of such therapeutic compounds,

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule which encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2; and b) a nucleic acid moleculewhich encodes at least 15 contiguous amino acids of SEQ ID NO:2.
 2. Anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of: a) the nucleotide sequence of SEQ ID NO:1;b) the nucleotide sequence of SEQ ID NO:1; wherein all T nucleotides arereplaced by U nucleotides; c) a nucleotide sequence complementary to (a)or (b); and d) a fragment of (a), (b), or (c) that is at least 25nucleotides in length.
 3. An isolated nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule which encodes apolypeptide that is at least 80% identical to SEQ ID NO:2; b) a nucleicacid molecule which hybridizes under stringent conditions to a nucleicacid molecule having the sequence of SEQ ID NO:1; and c) a nucleic acidmolecule which hybridizes under stringent conditions to a nucleic acidhaving the cDNA sequence contained within ATCC Accession No. ______. 4.A substantially pure polypeptide selected from the group consisting of:a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; andb) a polypeptide comprising at least 15 contiguous amino acids of SEQ IDNO:2.
 5. The polypeptide of claim 4, wherein the polypeptide is fused toa heterologous polypeptide.
 6. A substantially pure polypeptide selectedfrom the group consisting of: a) a polypeptide encoded by a nucleic acidmolecule which hybridizes under stringent conditions to the nucleic acidmolecule of SEQ ID NO:1; b) a polypeptide encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the cDNA sequencecontained within ATCC Accession No. ______.
 7. The polypeptide of claim6, wherein the polypeptide is fused to a heterologous polypeptide.
 8. Amethod for detecting the presence of a nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2; b) anucleic acid molecule which encodes at least 15 contiguous amino acidsof SEQ ID NO:2; in a sample, the method comprising the steps of: i)contacting the sample with a nucleic acid probe which selectivelyhybridizes to the nucleic acid molecule; and ii) determining whether thenucleic acid probe binds to the nucleic acid molecule in the sample. 9.The method of claim 8, wherein the sample comprises mRNA.
 10. A methodfor producing a substantially pure polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:2; and b) a polypeptide comprising at least 15 contiguousamino acids of SEQ ID NO:2; the method comprising the step of culturinga host cell containing the nucleic acid molecule encoding thepolypeptide under conditions in which the nucleic acid molecule isexpressed.
 11. The method of claim 10, wherein the host cell is abacterium.
 12. A method for detecting the presence of a polypeptideselected from the group consisting of: a) a polypeptide comprising theamino acid sequence of SEQ ID NO:2; and b) a polypeptide comprising atleast 15 contiguous amino acids of SEQ ID NO:2; in a biological sample,the method comprising the steps of: i) contacting the sample with acompound which selectively binds to the polypeptide; and ii) determiningwhether the compound binds to the polypeptide in the sample.
 13. Themethod of claim 12, wherein the compound which binds to the polypeptideis an antibody.
 14. A method for identifying a compound which binds tc apolypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; and b) a polypeptidecomprising at least 15 contiguous amino acids of SEQ ID NO:2; the methodcomprising the steps of: i) contacting the polypeptide with a testcompound; and ii) determining whether the polypeptide binds to the testcompound.
 15. The method of claim 14, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) direct detection of the binding; and b)detection of a competitor molecule which disrupts binding of the testcompound to the polypeptide.
 16. A method for modulating the activity ofa polypeptide selected from: a) a polypeptide comprising having aminoacid sequence of SEQ ID NO:2; and b) a polypeptide comprising at least15 contiguous amino acids of SEQ ID NO:2; the method comprisingcontacting a cell expressing the polypeptide with a compound which bindsto the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.
 17. The method of claim 16, wherein theactivity is recruitment of a caspase.
 18. The method of claim 16,wherein the method results in an increase in hexose uptake by the cell.19. The method of claim 16, wherein the method results in a decrease inhexose uptake by the cell.
 20. A method of identifying a compound thatmodulates the expression of a gene encoding GLUTX, the method comprisingthe steps of: a) contacting a cell expressing a gene with a testcompound; and b) detecting the level of expression of the gene in thepresence of the test compound, wherein a difference in expression in thepresence of the test compound compared to expression in the absence ofthe test compound indicates that the test compound modulates expressionof the gene.
 21. The method of claim 20, wherein the compound isselected from the group consisting of polypeptides, ribonucleic acids,small molecules, ribozymes, antisense oligonucleotide, anddeoxyribonucleic acids.
 22. A method of identifying a compound thatmodulates the activity of GLUTX, the method comprising the steps of: a)contacting the polypeptide with a test compound; and b) detecting thelevel of activity of GLUTX having the amino acid sequence of SEQ ID NO:2in the presence of the test compound, wherein a difference in activityin the presence of the test compound compared to the activity in theabsence of the test compound indicates that the test compound modulatesthe activity of GLUTX.
 23. The method of claim 22, wherein the compoundis selected from the group consisting of polypeptides, ribonucleicacids, small molecules, ribozymes, antisense oligonucleotides, anddeoxyribonucleic acids.
 24. A method for modulating hexose uptake, themethod comprising modulating the expression or activity of a geneencoding the amino acid sequence of SEQ ID NO:2.
 25. A method fortreating a patient having a disorder associated with aberrant expressionor activity of a gene encoding the amino acid sequence of SEQ ID NO:2,the method comprising administering a therapeutically effective amountof a compound that decreases the expression or activity of the gene. 26.The method of claim 25, wherein the compound is selected from the groupconsisting of polypeptides. ribonucleic acids, small molecules,ribozymes, antisense oligonucleotides, and deoxyribonucleic acids.
 27. Amethod for treating a patient having a disorder associated with aberrantexpression or activity of a GLUTX polypeptide comprising the amino acidsequence of SEQ ID NO:2, the method comprising administering atherapeutically effective amount of a compound that increases theexpression or activity of the gene.
 28. The method of claim 27, whereinthe compound is selected from the group consisting of polypeptides,ribonucleic acids, small molecules, ribozymes, antisenseoligonucleotides, and deoxyribonucleic acids.
 29. A method fordiagnosing a patient as having a disorder associated with aberrantexpression of GLUTX, comprising measuring expression of a GLUTXpolypeptide having the sequence of SEQ ID NO:2 in a biological sampleobtained from the patient, wherein increased or decreased GLUTXexpression in the biological sample, compared with GLUTX expression in acontrol sample, indicates that the patient has a disorder associatedwith aberrant expression of GLUTX.
 30. A method for diagnosing a patientas having a disorder associated with expression of an isoform of GLUTX,comprising isolating GLUTX mRNA or GLUTX polypeptide from the patientand determining the sequence of the mRNA or polypeptide, a difference inthe sequence, as compared to the nucleotide sequence of SEQ ID NO:l orthe polypeptide sequence of SEQ ID NO:2, respectively, indicatingexpression of an isoform of GLUTX.
 31. A method for diagnosing a patientas having a disorder associated with aberrant activity of GLUTX,comprising measuring the activity of a GLUTX polypeptide having theamino acid sequence of SEQ ID NO:2 in a biological sample obtained fromthe patient, wherein increased or decreased GLUTX activity in thebiological sample, compared with GLUTX activity in a control sample,indicates that the patient has a disorder associated with aberrantactivity of GLUTX.
 32. The method of claim 20, wherein the gene furthercomprises a sequence encoding an amino acid sequence selected from thegroup consisting of: i) the amino acid sequence of SEQ ID NO:2, and ii)at least 15 contiguous amino acids of SEQ ID NO:2.